Uracil DNA Glycosylase
I-Uracil-DNA Glycosylase (UNG okanye i-UDG) i-clone edibeneyo ye-E.coli ene-molecular weight 25 kDa.Ikhuthaza ukukhutshwa kwe-uracil yamahhala kwi-uracil equkethe i-DNA enye kunye ne-double-stranded DNA, kwaye ayisebenzi ngokuchasene ne-RNA, kwaye ingasetyenziselwa ukuthintela ukungcoliswa kweemveliso ze-PCR zokukhulisa.Umgaqo-nkqubo wesenzo usekelwe kwinto yokuba ukuba i-dUTP ifakwe endaweni ye-dTTP kwi-PCR reaction kwaye imveliso ye-PCR yokukhulisa equlethe iziseko ze-dU yenziwe, i-enzyme inokwaphula ngokukhethiweyo ibhondi ye-glycosidic ye-U iziseko kwi-single-stranded kunye ne-double-stranded. DNA kunye nokwehlisa umgangatho wemveliso ye-PCR yokukhulisa.
Isicelo esicetyiswayo
Ukwandiswa koThintelo loNgcoliseko
Imeko yoGcino
-20°C kwindawo yokugcina ixesha elide, kufuneka ixutywe kakuhle phambi kokusetyenziswa, kunqande ukunyibilika komkhenkce rhoqo.
Isithinteli sogcino
20 mM Tris-HCl (pH 8.0) , 150 mM NaCl, 1 mM EDTA, 1 mM DTT, Stabilizer, 50% Glycerol.
Inkcazelo yeyunithi
Ubungakanani be-enzyme efunekayo ukuthobisa i-1µg ye-DNA enemisonto enye equlethe iziseko ze-dU kwiyure ye-1 kwi-37 ° C yiyunithi ye-1.
Ulawulo lwemeko
1.I-SDS-PAGE ukucoceka kwe-electrophoretic ngaphezulu kwe-98%
2.Ukwandisa ubuntununtunu, i-batch-to-batch control, uzinzo
3.Emva kokuba i-1U ye-UNG iphathwe kwi-50 ℃ ye-2mins, ithempleyithi equlethe u-U ngaphantsi kweekopi ezili-103 kufuneka ithotywe ngokupheleleyo kwaye akukho mveliso yokukhulisa inokuveliswa.
4.Akukho msebenzi we-nuclease wangaphandle
Imiyalelo
| Amacandelo | Umthamo (μL) | Ugxininiso lokugqibela |
| 10 × PCR Buffer (dNTP simahla, Mg²+simahla) | 5 | 1× |
| ii-dUTPs (dCTP, dGTP, dATP) | - | 200 μM |
| I-dUTP (buyisela i-dTTP) | - | 200-600 μM |
| 25 mM MgCl2 | 2-8 μL | 1-4 mm |
| 5 U/μL Taq | 0.25 | 1.25 U |
| 5 U/μL I-UNG | 0.25 (0.1-0.5) | 0.25 U (0.1-0.5) |
| 25 × I-Primer Mixa | 2 | 1× |
| Isifanekiso | - | ＜1μg/impendulo |
| ddH₂O | Ukuya kwi50 | - |
Phawula: a: Ukuba isetyenziselwa i-qPCR/qRT-PCR, iprobe yefluorescent kufuneka yongezwe kwindlela yokusabela.Ngokuqhelekileyo, i-primer concentration yokugqibela ye-0.2 μM inokunika iziphumo ezilungileyo;xa i-reaction performance ihlwempuzekile, i-primer concentration inokulungiswa kuluhlu lwe-0.2-1 μM.Ngokuqhelekileyo, i-probe concentration ilungiselelwe kuluhlu lwe-0.1-0.3 μM.Imifuniselo yegradient yoxinaniso inokwenziwa ukufumana eyona ndibaniselwano yeprimer kunye neprobe.
Amanqaku
1.I-enzyme ye-UNG ingasetyenziselwa ukususa iimveliso zokukhulisa i-dUTP ezingcolisekileyo kwinkqubo yokusabela phambi kwe-PCR ye-amplification reaction, emva koko ukuphepha iziphumo zobuxoki ngenxa yokungcoliswa kwemveliso.
2.Ubushushu obufanelekileyo be-UNG enzyme ekufuneka isetyenziswe kwi-anti-contamination PCR reaction is generally 50℃ for 2mins;imeko yokungasebenzi yi-95℃ kwi-5mins.
3.Kuphephe ukunyibilika komkhenkce rhoqo, kwaye ungabi sesichengeni kuguquko olukhulu lobushushu.
4.Izakhi zofuzo ezahlukeneyo eziza kwandiswa zineendlela ezahlukeneyo zokusetyenziswa kwe-dUTP kunye nobuntununtunu kwi-enzyme ye-UNG, ke ngoko, ukuba ukusetyenziswa kwenkqubo ye-UNG kukhokelela ekunciphiseni uvakalelo lokubona, inkqubo yokusabela kufuneka ihlengahlengiswe kwaye iphuculwe, ukuba ufuna inkxaso yobugcisa, nceda uqhagamshelane. inkampani yethu.
